Herpesvirus infection is a major public health problem. Herpes simplex virus-1 (HSV-1), as the model virus for a-herpesvirus family, is the leading pathogen for infection-caused blindness and encephalitis. To establish infection and escape critical components of the immune systems to remain latent in host cells, the virus has evolved sophisticated immune evasion strategies. CD1d-restricted innate-like NKT cells play critical anti-microbial functions against different pathogens, including viruses, bacteria, fungi, and parasites. However, it remains unclear how pathogens including viruses evade the potent anti-microbial functions of NKT cells during infection and latency. The specific emphasis of this proposal is how HSV-1 modulates human CD1d expression in antigen-presenting cells to inhibit NKT cell function. We have originally discovered that upon infection, HSV-1 rapidly suppresses CD1d expression on the cell surface and inhibits the activation of NKT cells and their immunoregulatory function. Recently, we have identified that two viral factors, glycoprotein B (gB) and protein kinase US3, are required for optimal downregulation of CD1d surface expression. In particular, gB targets and relocates CD1d molecules to the TGN, while US3 blocks CD1d recycling and facilitates more gB-mediated CD1d relocalization to the TGN. Remarkably, this immune evasion mechanism only targets human, but not mouse, CD1d suggesting it is a result of viral co- evolution with human host. To dissect the in vivo roles of tis immune evasion mechanism, we have successfully generated a human CD1d knock-in mouse. We propose three interrelated specific aims. First, we will delineate the molecular details of the gB-CD1d interaction and map the interaction domains in these proteins. Second, we will examine the molecular and cellular mechanism by which the viral protein US3 cooperates with gB to down-regulate CD1d. Finally, we will investigate how gB and US3 proteins inhibit NKT cell function using our new mouse model. We will specifically examine how the down-regulation of CD1d inhibits the initial activation and recruitment of NKT cells to the infection site. Further identification of the NKT cell effector function inhibited by the evasins will provide novel leads or therapeutic means to treat and prevent HSV-1 infection. This proposal is highly innovative and its successful outcome should serve as a major discovery that will significantly impact our understanding of herpesvirus-mediated immune evasion as well as potentially provide the means for developing effective vaccines and antiviral drugs. PUBLIC HEALTH RELEVANCE: Herpes simplex virus infection is a major public health concern. This application proposes to investigate how herpes simplex virus-1, a prevalent human herpes virus, suppresses host immune system, particularly the innate CD1d/NKT system, to establish infection and latency. Understanding the pathogenesis of this virus will provide novel targets to design antivirals and new approaches to improve the efficiency of viral vaccines.